EP0621074B1 - Mit hydrophilem Polymer überzogene auf Basis von perfluorokohlenstoffhaltendem Polymer Matrisen, deren Herstellung und Verwendung in Bioaffinitätstrennungen - Google Patents

Mit hydrophilem Polymer überzogene auf Basis von perfluorokohlenstoffhaltendem Polymer Matrisen, deren Herstellung und Verwendung in Bioaffinitätstrennungen Download PDF

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EP0621074B1
EP0621074B1 EP94105965A EP94105965A EP0621074B1 EP 0621074 B1 EP0621074 B1 EP 0621074B1 EP 94105965 A EP94105965 A EP 94105965A EP 94105965 A EP94105965 A EP 94105965A EP 0621074 B1 EP0621074 B1 EP 0621074B1
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ligand
hydrophilic polymer
binder
perfluorocarbon
carrier
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EP0621074A1 (de
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Norman A. Parris
Christopher Robin Lowe
Ian Pitfield
Ducan Ross Purvis
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EIDP Inc
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EI Du Pont de Nemours and Co
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B01J20/267—Cross-linked polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/327—Polymers obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B01J20/3282—Crosslinked polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
    • B01J2220/49—Materials comprising an indicator, e.g. colour indicator, pH-indicator
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/814—Enzyme separation or purification
    • Y10S435/815—Enzyme separation or purification by sorption
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
    • Y10S530/815—Carrier is a synthetic polymer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
    • Y10S530/815—Carrier is a synthetic polymer
    • Y10S530/816—Attached to the carrier via a bridging agent

Definitions

  • This invention relates to inert solid affinity supports exhibiting low nonspecific binding, comprising perfluorocarbon polymer-based matrices having a coating of a crosslinked hydrophilic polymer such as polyvinyl alcohol and their use in affinity separations.
  • wetting agents such as surfactants and water miscible organic solvents will typically reduce adsorption, the effect is generally temporary, particularly where the surface is exposed to air. Further, the presence of these agents in solution can lead to foaming and negatively impact biological activity by disturbing the tertiary structure of proteins.
  • hydrophilic materials As supports for membranes or chromatographic processes. Materials such as cellulose, acrylamide, polyethyleneimine and agarose are widely used for this reason. Although possessing the desired low level of hydrophobic character, these naturally hydrophilic substances can be deficient in other ways. For example, many of these hydrophilic substances exhibit low, but finite, solubility in aqueous solutions. This is a concern in most biological process operations which generally utilize large volumes of aqueous fluids. Such solubilization leads to both physical breakdown of the support matrix and additionally, the potential for contamination of the end product by the foreign materials released from the support matrix. The presence of such contaminants in therapeutic agents is a major concern in commercial biotechnology as they may cause fever or initiate an unexpected immune response in the patient.
  • hydrophilic materials also exhibit limited thermal and chemical stability, thus interfering with their use in procedures for sterilization and endotoxin removal.
  • hydrophobic materials include polyethylene, polytetrafluoroethylene, refractory alumina and glass. While these materials generally possess good thermal and chemical stability, they are of limited use in biotechnology operations due to undesirable adsorption and nonspecific binding of biomolecules to their surfaces.
  • Affinity separations are generally considered to require the use of solid carriers derivatized with a ligand or binder.
  • Affinity chromatography is well known and has been reviewed, for example, in C. R. Lowe, "An Introduction to Affinity Chromatography, North holland Publishing Company, Amsterdam, New York, 1978. The list of support materials suitable for affinity chromatography is extensive and will not be reviewed here (see Lowe, 1978, for a partial listing).
  • Fluorocarbon polymers have been used as carriers to which ligands have been attached by adsorption [U.S. Pat. No. 3,843,443 issued to Fishman on Oct. 22, 1974; WO 8603 840 A filed by Rijsk Univ. Groningen; and Siergiej, Dissertation Abstracts, It. B., Volume 44, 153 (1983)].
  • Sakagani et al. [EP 0,011,504, published July 20, 1983], disclose the use of electrodeposition to attach ligands to fluoropolymer ion-exchange membranes.
  • U.S. Pat. No. 4,885,250 issued to Eveleigh et al. on Dec. 5, 1989 discloses a solid support based on an inert perfluorocarbon polymer carrier with perfluorocarbon-substituted ligands or binders attached to its surface.
  • U.S. Pat. No. 4,954,444 issued to Eveleigh et al. on Sep. 4, 1990 disclose a solid support based on an inert perfluorocarbon polymer carrier with ligands or binders for the ligands attached to its surface through a highly fluorinated isocyanate group.
  • Hato et al. U.S. Pat. No. 4,619,897, issued Oct. 23, 1986 disclose the immobilization of enzymes onto a fluorine resin membrane which is made hydrophilic on one side by the penetration of a perfluoralkyl surface active agent to a prescribed depth.
  • the asymmetrically functional membrane thus obtained is then treated with an enzyme and a crosslinking agent such as glutaraldehyde to achieve enzyme immobilization.
  • Murakami (Unexamined Patent Application Publ. [Kokai] No.: 64-38,448, laid open to the public on Feb. 8, 1989) discloses a method for impregnating the pores of a fluoroplastic polymer with a hydrophilic polymer and then crosslinking the hydrophilic polymer by irradiation with ultraviolet rays.
  • the support matrix used in this invention is thus suitable for use in various biotechnology operations and apparatus such as for example, support or separation membranes, affinity chromatography supports, ion exchange separations, and enzyme or cellular supports.
  • crosslinked hydrophilic polymer coated perfluorocarbon polymer-based matrix used in this invention can be used alone to minimize nonspecific binding of biological molecules, or, can be activated to covalently bind to biologically active molecules or other molecules of biological interest, such as for example, ligands or binders for ligands.
  • This invention relates to a solid affinity support having an attached ligand or binder for the ligand comprising:
  • This invention also relates to a process for preparing a solid support containing an attached ligand or binder for the ligand comprising the steps of:
  • Yet another aspect of this invention relates to a bioaffinity separation process comprising the steps of:
  • the instant invention is based on the surprising and unexpected finding that when a hydrophilic polymer, such as polyvinyl alcohol is coated or adsorbed onto a perfluorocarbon polymer-based carrier and subsequently crosslinked using a bifunctional crosslinking agent, the resulting hydrophilic coating is sufficiently strong to provide for a stable matrix which can withstand the various washing steps and other operations typically employed in various biotechnology applications, such as bioaffinity separations using membranes and affinity chromatographic separations.
  • the matrices and supports of the present invention are stable in aqueous environments and exhibit low nonspecific binding to proteins, nucleic acids, and other components of biological samples to surfaces.
  • perfluorocarbon is meant a molecule which contains the largest possible or a relatively large proportion of fluorine atoms in its structure.
  • Perfluorocarbon polymers are known to be inert.
  • Some perfluorocarbon polymers which can be used for the solid affinity supports of this invention are various Teflon® fluorocarbon polymers, polytetrafluoroethylene, polyvinylfluoride, and polyvinylidene difluoride. (Teflon® is a registered trademark of E. I. du Pont de Nemours and Company).
  • hydrophilic polymer an uncharged, hydrophilic, water soluble non-cyclic polymer having a multiplicity of hydroxyl groups sufficient for crosslinking the polymer molecules to adjacent like molecules (intermolecular crosslinking) so that the crosslinked hydrophilic polymer coating on the perfluorocarbon carrier is sufficiently strong and chemically stable to withstand the various operations and operating conditions typical of biotechnology processing steps, such as various washing steps used in affinity and membrane separations.
  • the hydrophilic polymer has at least one hydroxyl group, such as a primary or secondary hydroxyl group, available for crosslinking for every six carbons atoms per polymeric unit.
  • the hydrophilic polymer preferably has at least one site, such as a terminal hydroxyl group, available for binding to a ligand or binder for the ligand.
  • the hydrophilic polymer is a straight chain hydrophilic polymer having one hydroxyl group for every three or fewer carbon atoms per polymeric unit.
  • Polyvinyl alcohol having a molecular weight of from around 8,000 to around 15,000 is particularly preferred.
  • Polymers which are not useful as hydrophilic polymers include agarose, dextran, polyethylene glycol, polyethyleneimine, and starch. The molecular weight range which can be used for the hydrophilic polymer is 1,000 to the point of insolubility in water, generally around 20,000.
  • Polyvinyl alcohol is the preferred hydrophilic polymer which can be used as a coating for the perfluorocarbon carrier used to prepare the matrix and support of the present invention.
  • Polyvinyl alcohol is based on the repeating polymeric structure where n is the number of repeating polymeric units.
  • bifunctional crosslinking agent is meant a compound having sites capable of covalently binding with the hydroxyl groups of the hydrophilic polymer to effect an intermolecular crosslinking of the hydrophilic polymer molecules.
  • sites which are capable of reacting with the hydroxyl group include -COCl, COBr, -NCO, and CHO.
  • bifunctional is meant the presence of two sites on the crosslinking agent which can react with the hydroxyl groups of the hydrophilic polymer. In a homobifunctional crosslinking agent two sites on the crosslinking agent which can react with the hydroxyl groups of the hydrophilic polymer are the same.
  • Such crosslinking agents which can react with hydroxyl groups are well known (see for example, U.S. Pat. No.
  • Suitable crosslinking agents which can be used to crosslink PVA to itself include dialdehydes such as glutaraldehyde, and diisocyanates such as toluene diiisocyanate. Homobifunctional crosslinking agents are preferred and dialdehydes are the preferred crosslinking agents for PVA.
  • the hydrophilic polymer coated perfluorocarbon-based polymer matrix of the present invention can be used to prepare solid affinity supports having attached to their surfaces ligands or binders for the ligand; such supports are useful in performing affinity separations.
  • ligand an antigen, hapten, nucleic acid, enzyme substrate, vitamin, or other small organic molecule including enzyme effectors, and inhibitors
  • binder an antibody, enzyme, nucleic acid, binding protein, synthetic mimics of binding proteins such as polylysine and polyethyleneimines or other biomolecules capable of specific binding, enzyme/substrate etc. interactions.
  • the method for preparing the hydrophilic polymer coated perfluorocarbon polymer-based matrix of the present invention involves adsorbing or coating the hydrophilic polymer onto the surface of the perfluorocarbon carrier.
  • the perfluorocarbon carrier is initially wetted with a water miscible organic solvent such as acetone or tetrahydrofuran (THF).
  • THF tetrahydrofuran
  • the hydrophilic polymer, such as polyvinyl alcohol is then be mixed with the carrier in an amount sufficient to coat the carrier and adsorption allowed to proceed under controlled time, temperature and pH conditions.
  • a crosslinking agent is then added to allow the hydrophilic polymer to allow it to crosslink with like molecules.
  • a dialdehyde such as terephthaldehyde is used to crosslink the preferred hydrophilic polymer polyvinyl alcohol.
  • the crosslinking reaction is allowed to proceed under controlled time, temperature and pH conditions.
  • crosslinking agents and appropriate reaction conditions for their use are well known in the art (see for example, U.S. Pat. No. 4,101,380 issued July 18, 1978 to Rubinstein et al.).
  • the solid affinity support can be formed by activating the hydrophilic surface of the matrix under controlled time, temperature and pH conditions, so that the activated surface can covalently attach ligands or binders for the ligands.
  • activation methods are well known in the art. Examples of such activation methods are described by Stewart, D. J., Immobilization of Triazine Dyes On Inert Hydrophobic Supports For Affinity Chromatography, Thesis for the degree of Doctor of Philosophy, University of Cambridge, Kings College (1989).
  • Activation allows for the covalent attachment of any site on a ligand or binder for the ligand, such as the -NH 2 , or -C00H of proteins, via the hydroxyl groups, preferably the terminal hydroxyl groups, of the hydrophilic polymer coating.
  • a ligand or binder for the ligand such as the -NH 2 , or -C00H of proteins
  • the PVA can be activated by the addition of cyanuric chloride.
  • the ligand or binder for the ligand is attached to the solid hydrophilic polymer coated perfluorocarbon-based polymer matrix to form a solid affinity support by covalent attachment.
  • Means for covalently attaching ligands or binders for ligands to appropriately activated supports and means for optimizing such covalent attachment are known in the art. For example various methods for the covalent attachment of ligands methods are described by Stewart, D. J., Immobilization of Triazine Dyes On Inert Hydrophobic Supports For Affinity Chromatography, thesis for the degree of Doctor of Economics, University of Cambridge, Kings College (1989).
  • the solid hydrophilic coated perfluorocarbon polymer-based matrix used in the present invention can be used in a wide variety of applications.
  • the matrix can be used as a substrate for perfluorocarbon-based polymer electronic circuit boards.
  • a perfluorocarbon based polymer can be coated with a hydrophilic polymer such as PVA and treated so as to deposit various conductive materials, such as metals on its surfaces.
  • PVA can be adsorbed onto a perfluorocarbon polymer-based support which has been previously treated with photo-resist and exposed to ultraviolet light, so as to define desired electrical paths.
  • the PVA coated perfluorocarbon matrix can be dipped into a solution of dissolved silver nitrate (the silver nitrate can be dissolved by the addition of dilute ammonia), and a few drops of a reducing agent such as formaldehyde added, until the silver is deposited in the support.
  • a reducing agent such as formaldehyde added
  • the matrix used in the present invention can also be used for various applications as wettable polymeric films or powders.
  • the supports of the present invention can also be used in immunoassays.
  • One such assay is a qualitative enzyme linked immunosorbent assay (ELISA) in which color can be visually detected on the surface of filter paper or other surfaces. Detectable signals other than color can also be used.
  • ELISA enzyme linked immunosorbent assay
  • an enzyme can be bound to a perfluorocarbon membrane of an electrochemical gas sensor.
  • the enzyme is chosen so as to be able to catalyze a reaction which generates a product or consumes coreactant which can be monitored electrochemically.
  • the electrochemical signal provides a measure of the analyze concentration.
  • the enzyme acts as the binder and the target analyze as the ligand.
  • perfluorocarbon particles (6-8 m 2 /g, mean particle size 70 microns obtained from E. I. Du Pont de Nemours and Company, Wilmington, De.) were stirred overnight in 500 ml tetrahydrofuran and then washed in 500 ml acetone on a glass sinter (grade 2) filter. The remaining acetone was drained under gravity leaving the particles still wet with acetone and unexposed to air.
  • the resulting translucent material was added to a stirred solution of aqueous 0.7 mM polyvinyl alcohol (PVA) (MW 14,000, 100% hydrolyzed) (Aldrich Co., Gillingham, Dorset, UK) and the PVA was allowed to adsorb for 5 hours at 20 degrees C, after which time 50 ml of 70mM aqueous terephthaldehyde (Aldrich, Gillingham, Dorset, UK) was added.
  • PVA polyvinyl alcohol
  • the mixture was acidified by the addition of 20 ml of 5 M HCl and after 4 hours of crosslinking, the material allowed to settle and the supernatant decanted off.
  • the resulting crosslinked polyvinyl alcohol coated perfluorocarbon matrix was washed on a sintered glass filter consecutively with 2 liters of water, 2 liters of hot water at 60 degrees C, 2 liters) and 2 liters of distilled water.
  • the amount of PVA adsorbed to the support was determined by difference analysis of the supernatant using a specific PVA assay described by Zwick, M.M.. J. Appl. Polm. Sci., 9,p. 2393 (1965), hereby incorporated by reference. It was found to be approximately 20 mg PVA per 1 g perfluorocarbon carrier.
  • Samples of 0.5 grams (g) crosslinked PVA coated perfluorocarbon polymer-based matrix having 20 mg PVA/g perfluorocarbon carrier and prepared as described above was incubated in a series of aqueous sodium hydroxide (NaOH) solutions of 0.0, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0M for 1 hour.
  • NaOH sodium hydroxide
  • the resulting material was then filtered using a sintered glass filter and added to 5 milliliters (ml) of 20mM cyanuric chloride (Aldrich, Gillingham, Dorset, UK) in acetone for 10 minutes at 20 degrees C.
  • the resulting activated material was washed consecutively, avoiding exposure to air, with acetone, acetone/water (50:50 v/v) and water prior to evaluating the number of reactive groups coupled.
  • the extent of activation ( ⁇ m Cl-/g activated material) was determined by hydrolyzing 0.1 g of reactive material in 3 ml of 0.1 M NaOH for 1 hour at 20°C, and assaying the chloride ions liberated using a method described by Vogel, A. I., Textbook of Quantitative Inorganic Analysis", 1978, Longman Inc., N.Y., pp.754, hereby incorporated by reference.
  • Figure 1 is a graph which shows that a concentration of 1 M NaOH in the prewash was optimal for introducing reactive cyanuric chloride groups into the matrix. Reactivity of the activated material fell at higher concentrations of alkali because stronger base either hydrolyzed the coupled cyanuric chloride or caused more extensive cross-linking of the PVA coating. Conversely, the use of a lower concentration of NaOH reduced the hydrolysis of cyanuric chloride groups on the adsorbent and thus minimized the introduction of possible non-specific adsorption sites.
  • the resulting activated material was washed consecutively, avoiding exposure to air, with acetone, acetone/water (50:50 v/v) and water prior to evaluating the number of reactive groups coupled.
  • the extent of activation ( ⁇ m Cl-/g activated material) was determined as described above.
  • Figure 2 is a graph which shows the effect on the reactivity of the material of increasing the cyanuric chloride concentration while using 1M NaOH as the optimum prewash condition.
  • the extent of activation increased linearly to a pseudo-plateau at approximately 70 ⁇ m Cl-/g, demonstrating that the level of reactivity of the support could be easily controlled.
  • a concentration of 10 mM cyanuric chloride produced a support containing 20 ⁇ m Cl-/g material.
  • Figure 3 is a graph which shows the stability of the activated material under various conditions.
  • One gram of reactive material of 20 ⁇ m Cl-/g activated material prepared as described above was stored at 20 degrees under three different conditions: in 0.1M NaOH, in 0.1 M sodium phosphate buffer pH 7.0, and 0.1 M acetic acid.
  • the coupled reactive groups were immediately hydrolyzed in 0.1 M NaOH but retained at least half of their reactivity in 0.1 M acetic acid and 0.1 M sodium phosphate buffer pH 7.0 for up to one week.
  • a sample of the activated material prepared as described above was stored at 4° C, after freeze drying in 10% (v/v) acetic acid and showed no apparent loss of reactivity, as measured using the procedure described herein, after 3 months of storage.
  • HSA human serum albumun
  • the amount of protein was determined with the Pierce Coomassie protein assay reagent (1.0 ml.) (Pierce, Luton, Beds, UK) added to appropriate serial dilutions of the protein solution (20 ⁇ l). After mixing and standing at room temperature for 10 minutes, the absorbance at 595 nm was measured. Standard curves for human serum albumin (HSA), human immunoglobulin G (IgG) (donated by PMLS, Porton Down, Wiltshire, UK) and concanavalin A (Con A) were prepared.
  • HSA human serum albumin
  • IgG human immunoglobulin G
  • Con A concanavalin A
  • Protein concentrations in stock solutions were initially determined by absorbance at 280 nm, assuming A280 nm 1% (w/v) 5.8, 14.7, (Nakamura, K., Hashimoto, T., Kato, Y., Shimuran, K. and Kasai, K.-I., J. Chromatogr., 510 (1990) 101.) and 1.1 (Borchert, A., Larsson, P.-O. and Mosbach, K., J. Chromatogr., vol. 244, 1982, p. 49.) for HSA, IgG and Con A, respectively and A275 nm 1% (w/v) was 1.65 for Protein A (Langone, J. J., Adv. Immunol., vol. 32, 1982, 157).
  • Figure 4 is a graph which shows the coupling of human serum albumin (HSA) to the activated support at different pH values.
  • HSA human serum albumin
  • FIG. 5 is a graph which shows the results of similar studies with the coupling of IgG at pH values 4-11.
  • the same coupling conditions were used as previously described for the coupling of HSA.
  • a broad optimum capacity of 10 mg. IgG/g material was obtained in the pH range 5-8.
  • immobilization of IgG at higher pH values, especially when compared to HSA was probably related to decreased protein solubility, which encouraged interaction with the reactive support prior to solvolysis.
  • Hydrolysis of the activated support with ethanolamine under the same conditions described above with respect to HSA reduced the amount of IgG coupled, but not to the very low levels observed for adsorption of protein to PVA-coated perfluorocarbon polymer-based matrix prepared as previously described herein.
  • Figure 6 is a graph which shows that at pH 5.0 and 20° C, approximately 60% of added albumin is coupled to the activated support within 5-10 min. with maximum immobilization achieved after 2 hours.
  • An amount of 0.2 g of activated material was separately added to samples of HSA (4mg/2ml) in 0.1 M acetate buffer at pH 5.0. Coupling was terminated at time intervals and the amount of immobilized or attached HSA was determined by assay of the supernatant.
  • the high reactivity of the active cyanuric chloride groups facilitates rapid immobilization of biochemicals to the support in aqueous media and at physiological pH.
  • Figure 7 is a graph which shows that HSA, IgG and concanavalin A all exhibit similar isotherms (2ml solutions of increasing concentrations were incubated with cyanuric activated material [0.2 g, 20 ⁇ mol Cl-/g) in 0.1 M acetate buffer pH 5.0 for 2 hours] with optimum coupling of approximately 8-10 mg./g. activated material and coupling yields of approximately 80%, even at relatively low protein concentrations. All of the proteins were immobilized with a surface coverage of approximately 1.4 mg./m 2 ., a figure characteristic for protein adsorption (see for example, Andrade, J. D. and Hlady, V., Adv. Polym. Sci., vol. 79, 1986, 12).
  • the solid affinity supports as prepared below were equilibrated in a relevant buffer and packed in Pharmacia HR 5/10 columns at a flow rate of 5 ml./min. and used in conjunction with a Pharmacia FPLC system comprising a P500 pump, LCC 500 plus controller, UV-I single path monitor and LKB 2212 Helirac fraction collector.
  • Concanavalin A (lectin) was coupled to the activated material and tested for their ability to purify horse radish peroxidase (HRP).
  • a concanavalin A affinity support was prepared by coupling the lectin in the presence of alpha-methyl-D-glucopyranoside (Sigma, Poole, Dorset, UK) in 0.5 M acetate, 0.5M NaCl, 1mM CaCl 2 , 1mM MnCl 2 , pH 5.1 (coupling buffer) for 2 hours. Before use the material was washed with coupling buffer and stirred in 0.1M ethanolamine pH 9.0 for 48 hours at 4° C.
  • a concanavalin A affinity support was synthesized by coupling the lectin in the presence of alpha-methyl-D-glucopyranoside in 0.05 M acetate, 0.5 M NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 , pH 5.1 for 2 h. Before use, the material was washed with coupling buffer and stirred in 0.1 M ethanolamine pH 9.0 for 48 h. at 4°C.
  • HRP horse radish peroxidase
  • Fig. 8A Concanavalin A affinity support
  • the RZ ratio (Absorbance at 405nm/Absorbance at 280nm) of the eluted fraction of 2.2 corresponded to a 5.6-fold purification of the crude material with 80% overall recovery.
  • the column employed was 0.5 X 10 cm, contained 3.6 mg Concanavalin A/ gram support, and 70 ⁇ m perfluorocarbon carrier.
  • Column conditions were as follows: mobile phase 0.05 M acetate, 0.5M NaCl, 1mM CaCl 2 , (FSA, Loughborough, Leics, UK) 1mM MnCl 2 , pH 5.1; flow rate: 1 ml/minute; sample was injected at 1 minute, 1ml crude HRP (5mg/ml); elution buffer: 25 mM alpha-methyl-D-glucopyranoside in mobile phase (3ml) injected at 5 minutes; fractions (1ml) were assayed at 280nm and 405nm.
  • Protein A purified from cell walls of Staphylococcus aureus , Cowan strain, Sigma Co., Poole, Dorset, UK
  • an cyanuric chloride activated perfluorocarbon polymer-based activated matrix having 4.6 ⁇ mol Cl-/g activated matrix using the coupling procedure described above.
  • plasma obtained from a known donor at the National Blood Transfusion Center, Nottingham, UK
  • the fraction eluted with 0.1 M citrate pH 3.0 contained 0.9 mg. IgG, a capacity equivalent to other supports (see for example, Fuglistaller, J. Immunol. Meth., vol. 124, 1989, p. 171).
  • the column employed was 0.5 X 10 cm, contained 0.7 mg Protein A/ gram support, and 70 micro-m perfluorocarbon carrier.
  • Column conditions were as follows: mobile phase 0.1 M Na2HPO4 buffer pH 8.0; flow rate: 2 ml/minute; sample was injected at 1 minute 1ml human plasma; elution buffer: 0.1M citrate pH 3.0 (3ml) injected at 6 minutes; fractions (2 ml) were assayed at 280nm.

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Claims (6)

  1. Fester Affinitätsträger mit einem gebundenen Liganden oder Bindemittel für den Liganden, umfassend:
    (a) ein chemisch inertes festes Substrat aus Perfluorkohlenstoff-Polymer;
    (b) eine vernetzte hydrophile Polymerbeschichtung auf dem Substrat; und
    (c) einen Liganden oder ein Bindemittel für den Liganden, der bzw. das an die Oberfläche des hydrophilen Polymers gebunden ist, wobei
    (i) der Ligand aus der Gruppe ausgewählt ist, die aus Nucleinsäure und Vitamin besteht,
    (ii) das Bindemittel für den Liganden aus der Gruppe ausgewählt ist, die aus Antikörper, Enzym und Nucleinsäure besteht.
  2. Träger gemäß Anspruch 1, wobei es sich bei dem hydrophilen Polymer um Polyvinylalkohol handelt.
  3. Träger gemäß Anspruch 1, wobei das Substrat aus der Gruppe ausgewählt ist, die aus Polytetrafluorethylen, Polyvinylfluorid und Polyvinylidendifluorid besteht.
  4. Verfahren zur Herstellung des festen Trägers gemäß Anspruch 1 bis 3, der einen gebundenen Liganden oder ein gebundenes Bindemittel für den Liganden enthält, umfassend die Schritte:
    (a) Adsorbieren eines hydrophilen Polymers an die Oberfläche eines Perfluorkohlenstoff-Substrats;
    (b) Vernetzen des hydrophilen Polymers unter Verwendung eines bifunktionellen Vernetzungsmittels;
    (c) Aktivieren der Oberfläche des vernetzten hydrophilen Polymers, das auf das Substrat auf Perfluorkohlenstoffbasis aufgetragen ist, so daß ein Ligand oder ein Bindemittel für den Liganden an seine Oberfläche gebunden werden kann; und
    (d) Binden eines Liganden oder eines Bindemittels für den Liganden an die Oberfläche des aktivierten hydrophilen Polymers, wobei
    (i) der Ligand aus der Gruppe ausgewählt ist, die aus Nucleinsäure und Vitamin besteht,
    (ii) das Bindemittel für den Liganden aus der Gruppe ausgewählt ist, die aus Antikörper, Enzym und Nucleinsäure besteht.
  5. Verfahren gemäß Anspruch 4, wobei das Vernetzungsmittel aus der Gruppe ausgewählt ist, die aus Dialdehyden und Diisocyanaten besteht.
  6. Bioaffinitäts-Trennverfahren, umfassend die Schritte:
    (A) Bilden eines festen Affinitätsträgers durch
    (a) Adsorbieren eines hydrophilen Polymers an die Oberfläche eines Perfluorkohlenstoff-Substrats;
    (b) Vernetzen des hydrophilen Polymers unter Verwendung eines bifunktionellen Vernetzungsmittels;
    (c) Aktivieren der Oberfläche des vernetzten hydrophilen Polymers, das auf das Substrat auf Perfluorkohlenstoffbasis aufgetragen ist, so daß ein Ligand oder ein Bindemittel für den Liganden an seine Oberfläche gebunden werden kann; und
    (d) Binden eines Liganden oder eines Bindemittels für den Liganden an die Oberfläche des aktivierten hydrophilen Polymers, wobei
    (i) der Ligand aus der Gruppe ausgewählt ist, die aus Nucleinsäure und Vitamin besteht,
    (ii) das Bindemittel für den Liganden aus der Gruppe ausgewählt ist, die aus Antikörper, Enzym und Nucleinsäure besteht; und
    (B) Abfangen eines Bindemittels oder eines Liganden für das Bindemittel, das bzw. der komplementär zu dem Liganden oder Bindemittel ist, der bzw. das an das Substrat gebunden ist, aus einem Gemisch unter Verwendung des festen Affinitätsträgers.
EP94105965A 1993-04-22 1994-04-18 Mit hydrophilem Polymer überzogene auf Basis von perfluorokohlenstoffhaltendem Polymer Matrisen, deren Herstellung und Verwendung in Bioaffinitätstrennungen Expired - Lifetime EP0621074B1 (de)

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US7163633B2 (en) 2001-02-01 2007-01-16 Sigma-Aldrich Co. Affinity matrices with enhanced visibility for molecular pull-down and immunoprecipitation applications
US7438806B2 (en) 2001-02-01 2008-10-21 Sigma-Aldrich Co. Affinity matrices with enhanced visibility for molecular pull-down and immunoprecipitation applications

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